Isolation, Culture And Functional Characterization Of Endothelial Progenitor Cells From Adults With Moyamoya Disease

Objective:1. To explore the experimental methods of angiogenesis in moyamoyadisease based on culture endothelial progenitor cells in vitro and characterizationobserved.2. To examine that whether these endothelial progenitor cells had normalproliferation, adhesion, migration ability compared with healthy control-group.Method:1. We obtained20ml of blood from6patients with MMD prior to their surgicaltreatment and from12healthy volunteers. All patients underwent conventionalangiography to confirm the diagnosis of MMD. Patients with moyamoya syndromeassociated with other cerebrovascular disease, blood diseases, immune system diseaseswere excluded.2. endothelialcells were generated in vitro from blood ormononuclearcells: Monoclear cells(MNCs) were isolated from peripheral blood by Ficolldensity gradient centrifugation, and then the cells were plated on fibronectin-coatedculture dishes. MNCs were cultured for7days by M199which contain vascularendothelial growth factor (VEGF) and Basic fibroblast growth factor(BFGF). Cellmorphology was observed under inverted phase contrast microscope. EPCs werecharacterized for Dil-acLDL and FITC-UEA-I by direct flurescent staining under a laserscanning conflcal microscope.3. Cell function tests: Determinating cell suspension’s ODvalue on3,6,9,12,15,18days by CCK8kits. EPCs migration ability on7days culturedwere tested by the scratch test method, the same cells were used to do adhesion assayto compare two groups of cell adhesion ability.Result:1.Peripheral blood mononuclear cells were isolated and cultured for3days,some cells formed irregularly, cells were cultured for7days, some cells gathered wereclumps or clusters installed, cells were cultured for14days, colony increased.2. Cells ofthe two groups had the fastest growth rate of9-12days, reached a plateau after the15days. The fastest growth rate of experimental group on9-12days is lower than controlgroup but is similar in the plateau. Experimental group EPCs number of migrating cells was26.67±2.33, in the control group was29.30±2.68, P=0.51, no difference betweenthem. The EPCs adhesion of the cells of the experimental group was22.17±2.68, in thecontrol group was20.60±2.18, P=0.85, the adhesion ability between the two groups issimilar.Conclusion:1. Moyamoya disease in adult patients with peripheral blood mononuclearcells can be successfully induced differentiation of endothelial progenitor cells.2.Compared with normal control group, EPCs from moyamoya patients have similargrowth pattern, morphology, proliferation, migration and adhesion ability.3. Moyamoyadisease vascular characteristic lesions may be related to the the pathological vascularenvironment in which a variety of regulatory factors, with normal differentiation of EPCsfunction in the body are affected, participate in or facilitate the process of moyamoyadisease pathology.4. Adult moyamoya disease in vivo reserves a strong vascular repairand regeneration potential, direct or indirect intracranial and extracranial vascularanastomosis in time may improve local blood flow induced more angiogenesis, toimprove the organization of regional cerebral ischemic symptoms.